The green tiger shrimp, P. (penaeus) semisulcatus is the most important of the Penaeid family in Persian Gulf. Based on morphological characters, the new subspecies P.semisulcatus persicus is recognized. In the present study, the mitochondrial D- loop and genomic ITS2 markers were used for the molecular comparison of P. semisulcatus and P.semisulcatus persicus. Three samples of the species and three samples of the subspecies were collected and muscle extracted genomic DNA was used as template. D-Loop and ITS2 were amplified and sequenced using 12S (D-loopF) /PCR-IR (D-loopR) and ITS2F/ITS2R Primers. Multiple sequences alignment and genetics distance were conducted using ClustalW2 and MEGA5.2. Results of sequence analysis showed that the D- loop and ITS2 are AT and GC rich, respectively. The sequence data analysis of the D-loop and ITS2, species and subspecies represent distinct clads, with sequence divergence of 31.9% - 32.5% in D- loop (613 -615 base) and 24.8% - 32.3% in ITS2 (335 base). Phylogenetic study reveals that Penaeus semisulcatus species and subspecies are more closely related to each other than to other phylogenetically related Penaeus species. The results from this study suggest the occurrence of two cryptic species in the Persian Gulf Shrimp, Penaeus semisulcatus.